Immunogenic peptide composition for the prevention and treatment of alzheimer&#39;s disease

ABSTRACT

The present invention relates to a composition comprising a peptide immunogen useful for the prevention and treatment of Alzheimer&#39;s Disease. More particularly, the peptide immunogen comprises a main functional/regulatory site, an N-terminal fragment of Amyloid β (Aβ) peptide linked to a helper T cell epitope (Th) having multiple class II MHC binding motifs. The peptide immunogen elicits a site-directed immune response against the main functional/regulatory site of the Aβ peptide and generate antibodies, which are highly cross-reactive to the soluble Aβ 1-42  peptide and the amyloid plaques formed in the brain of Alzheimer&#39;s Disease patients. The antibodies elicited being cross reactive to the soluble Aβ 1-42  peptide, promote fibril disaggregation and inhibit fibrillar aggregation leading to immunoneutralization of the “soluble Aβ-derived toxins”; and being cross-reactive to the amyloid plaques, accelerate the clearance of these plaques from the brain. Thus, the composition of the invention comprising the peptide immunogen is useful for the prevention and treatment of Alzheimer&#39;s Disease.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional application, which claims the benefitof U.S. patent application Ser. No. 10/861,614 entitled “ImmunogenicPeptide Composition Comprising A Promiscuous Helper T Cell Epitope AndAn N-Terminal Fragment Of ABeta₁₋₄₂ Peptide”, filed Jun. 4, 2004, whichis a divisional application of, and claims the benefit to, U.S. patentapplication Ser. No. 09/865,294 entitled “Immunogenic PeptideComposition Comprising Measles Virus F Protein T Helper Cell Epitope(MVF Th1-16) And N-Terminus Of B-Amyloid Peptide”, filed on May 25,2001, (now U.S. Pat. No. 6,906,169), all of which are hereinincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a composition comprising a peptideimmunogen useful for the prevention and treatment of Alzheimer'sDisease. More particularly, the peptide immunogen comprises a mainfunctional/regulatory site, an N-terminal fragment of Amyloid β (Aβ)peptide linked to a helper T cell epitope (Th) having multiple class IIMHC binding motifs. The peptide immunogen elicits a site-directed immuneresponse against the main functional/regulatory site of the Aβ peptideand generate antibodies, which are highly cross-reactive to the solubleAβ₁₋₄₂ peptide and the amyloid plaques formed in the brain ofAlzheimer's Disease patients. The antibodies elicited being crossreactive to the soluble Aβ₁₋₄₂ peptide, promote fibril disaggregationand inhibit fibrillar aggregation leading to immunoneutralization of the“soluble Aβ-derived toxins”; and being cross-reactive to the amyloidplaques, accelerate the clearance of these plaques from the brain. Thus,the composition of the invention comprising the peptide immunogen isuseful for the prevention and treatment of Alzheimer's Disease.

BACKGROUND OF THE INVENTION

Alzheimer's Disease (AD) is a chronic, neurodegenerative disordercharacterized by a loss of cognitive ability and severe behavioralabnormalities in a patient leading to the eventual death of the patient.There are currently 2.5 to 4.0 million AD patients in the U.S. and 17 to25 million worldwide. It is the fourth leading cause of death in Westerncultures, preceded only by heart disease, cancer, and stroke. ARICEPT®,an acetylcholinesterase inhibitor has been approved by the FDA fordecelerating the rate of decline of Alzheimer patients. However, it iseffective only for a limited period of time and in some patients. Up tothe present there is no definitive treatment or cure for thisdevastating disease.

Two microscopic deposits, i.e., neurofibrillary tangles (NFT) and senileamyloid plaques, were identified by Alois Alzheimer as the pathologichallmarks of the disease. The neurofibrillary tangles consist of two 10nm wide filaments twisted around each other, referred to as pairedhelical filaments (PHFs), a major component of which is phosphorylatedtau. The phosphorylation of serine at amino acid 262 of the tau proteinrepresents a crucial step leading to physiological dysfunction of tau.PHFs are intracellular and are found in many of the abnormal dendriticand axonal processes, or neurites that make up the periphery of senileamyloid plaques. The senile amyloid plaques consist of disorganizedneurophil filaments in an area of up to 150 μm in cross section with anextra-cellular core of amyloid deposit. The cerebral amyloid plaques areultrastructurally distinct from PHFs and consist of 4-8 nm widefilaments that are not wound together in pairs. The plaque core consistsof aggregates of a peptide, initially referred to as A4, with a relativemolecular mass (M) of about 4,000 (Masters et al., Proc Natl Acad SciUSA, 1985, 82:4245-4249).

A partial amino acid sequence of A4, now renamed amyloid β peptide (orAβ₁₋₄₂), shows that it is similar to the amyloid β protein isolated fromcerebral blood vessels of patients with Alzheimer's disease or Down'ssyndrome (Glenner and Wong, Biochem Biophys Res Comm, 1984; 120:885-890;122:1131-1135).

Aβ₁₋₄₂ has been hypothesized to be related to AD for a number ofreasons. Firstly, in peripheral amyloidoses, e.g., primary light chaindisease or secondary AA amyloidosis, large amyloid burdens stronglycorrelate with tissue and organ dysfunction. Secondly, amyloid plaquedensity positively correlates with premortem dementia scores in AD.Thirdly, Aβ₁₋₄₂ deposition is the earliest neuropathological marker inAD and related disorders such as Down's syndrome, where it can precedeNFT formation by 2-3 decades. Fourthly, β-amyloidosis is relativelyspecific to AD and related disorders. Fifthly, Aβ₁₋₄₂ is toxic toneurons (Yankner et al., Science, 1990; 250:279-282). Lastly, missensemutations in the structural amyloid precursor protein (APP) gene causeearly onset of familial AD (Goate et al., Nature, 1991; 349:704-706;Mullan et al., Nature Genetics, 1992; 1:345-347). Notably, one suchmutation causes dramatic Aβ₁₋₄₂ overproduction (Citron et al., Nature,1992; 360:672-674).

In 1987, Kang et al. (Nature, 1987; 325:733-737) and three other groups(see 1987 status reports by Anderton, Nature, 1987; 325:658-659 andBarnes, Science, 1987; 235:846-847) independently cloned the gene fromwhich Aβ₁₋₄₂ is derived. This gene, now known as the amyloid precursorprotein (APP), encodes a protein of 695 amino-acid residues with a MW ofabout 79,000 that is expressed in virtually all tissues. There are atleast five splicing variants of APP, four of which contain the β-amyloidpeptide sequence.

Four genes have been implicated in familial forms of AD. Three of thegenes, βAPP, presenilin I, and presenilin 2, when mutated, causeautosomal dominant early forms of AD. The fourth gene, Apolipoprotein E,has a naturally occurring polymorphic form, ApoE4, that represents amajor genetic risk factor for the development of the disease. Theconcept that alterations in several distinct genes can lead to a chronicimbalance between Aβ₁₋₄₂ production and its clearance, with theresulting aggregation of first the 42-residue and then the 40-residuepeptide into cytotoxic plaques, is supported by available evidence. Theevidence strongly suggests that defects in each of these four genespredispose the AD phenotype by (1) enhancing the production and/or thedeposition of Aβ₁₋₄₂ peptides or (2) by decreasing the clearance ofApoE4 from tissue (Selkoe, J Biol Chem, 1996; 271:18295-18298).

From available data, it appears that aggregated but not monomeric Aβ₁₋₄₂peptides can induce cell dysfunction and death in vitro by a range ofpresumably interrelated mechanisms. These include oxidative injury(Thomas et al., Nature, 1996; 380:168-171; Behl et al., Cell, 1994;77:817-827), alterations in intracellular calcium homeostasis (Arispe etal., Proc Natl Acad Sci USA, 1993; 90:567-571), and cytoskeletalreorganization (Busciglio et al., Neuron, 1995; 14:879-888). Sufficientknowledge of some of the principal steps in the amyloid-induced cascadehas emerged, even though the cascade hypothesis is hotly contested.

Pharmalogical approaches of identifying small molecules which couldinhibit one or another step of the amyloid induced cascade are now wellunder way. Of particular interest are two approaches: attempts tointerfere with the aggregation of Aβ₁₋₄₂ peptides by decreasing thesecretion of Aβ₁₋₄₂ peptides from neuronal and glial cells or inhibitthe toxicity that these extracellular aggregates produce on neurons andglial cells and their processes. A third approach which attempts tocontrol the specialized inflammatory response that appears to betriggered by aggregated Aβ₁₋₄₂ (including microglial stimulation,activation of the classical complement cascade, cytokine release, andreactive astrocytosis) may prove to be of benefit to Alzheimer'spatients.

Aside from the above-mentioned pharmacological approaches for ADintervention, immunological interventions have also been attempted.Soloman et al. (Proc Natl Acad. Sci, 1996; 93:452-455; Proc Natl Aca.Sci, 1997; 94:4109-4112) showed that three specific monoclonalantibodies, directed toward a site in the N-terminal region of the humanAβ₁₋₄₂ peptide, bind in varying degrees to preformed fibrils leading totheir disaggregation and inhibition of their neurotoxic effect. Theantibodies were also found to prevent the formation of fibrillar Aβ₁₋₄₂.Solomon et al. (WO 01/18169) also attempted to prepare a phage displayof an epitope of the Aβ₁₋₄₂ peptide and administering the phagedisplayed epitope or peptide containing the epitope intraperitonially tomice to elicit antibodies to the Aβ₁₋₄₂ peptide. In vitro testing withrat phenochromocytoma showed that a 1:5 dilution of the antiseraprevented the neurotoxicity of Aβ₁₋₄₂. The antiserum at a dilution of1:5 and 1:20 was also shown to disrupt the fibril structure of Aβ invitro with extensive deterioration of fibril morphology. However, theadjuvant used was for the first injection was Complete Freund's Adjuvantwith the incomplete Freund's Adjuvant for the second injection. Theadjuvants used are entirely unsuitable for use in humans. Moreover, thelevels of antibodies generated were too low to be effective despite theuse of these harsh adjuvants.

Subsequently, Schenk et al. (Nature, 1999; 400:173-177) showed thatimmunization with Aβ₁₋₄₂ peptide inhibits the formation of amyloidplaques and the associated dystrophic neurites in a mouse model of AD.However, due to the low immunogenicity of the Aβ₁₋₄₂ peptide, the methodemployed required repeated administrations of the antigen with a harshlesion-forming adjuvant to obtain the higher levels of anti-Aβ₁₋₄₂plaque antibodies necessary to affect plaque formation. Moreover, it wascautioned that immunization with Aβ₁₋₄₂ might induce more accumulationof the toxic amyloid itself (Araujo, D M & Cotman, C W, Brain Res, 1992;569, 141-145).

Despite these criticisms, additional studies in transgenic AD mousemodels through similar active immunization have lent credence to theimmunoprophylaxis and immunotherapeutic approaches for AD. Janus et al.(Nature, 2000; 408:979-982) described Aβ₁₋₄₂ peptide immunization in amouse model for AD that reduced behavior impairment and plaques. Morganet al. (Nature, 2000; 408:982-985) described Aβ₁₋₄₂ peptide vaccinationto prevent memory loss in the mouse model.

Direct support for the effectiveness of immune therapy came from theobservation that peripheral administration of antibodies, monoclonal orpolyclonal, against Aβ-peptide reduced amyloid burden (WO 99/27944; Bardet al., Nature Medicine, 2000; 6:916-919). Despite relatively modestserum levels, these passively administered antibodies, monoclonal 3D6(anti-Aβ₁₋₅) and 10D5 (anti-Aβ₁₋₁₂) or polyclonal anti-Aβ₁₋₄₂, were ableto enter the central nervous system. There, the antibodies bound toplaques and induced clearance of pre-existing amyloid plaques. Bard etal., reported that when examined in an ex vivo assay with brain sectionsof PDAPP mice (i.e., mice transgenic for an APP mini-gene driven by aplatelet-derived growth factor promoter) or AD patient brain tissue,antibodies against Aβ-peptide triggered microglial cells to clearplaques through Fc receptor-mediated phagocytosis and subsequent peptidedegradation. This study demonstrated that passively administeredantibodies against Aβ₁₋₄₂ peptide and the Aβ₁₋₄₂ N-terminus regionreduced the extent of plaque deposition in a mouse model of AD; and thatmonoclonal antibodies or polyclonal antibodies elicited by site-directedvaccines are able to enter the CNS at therapeutically relevant levels.

Despite the promising findings of immunological intervention in micemodel for AD, a vaccine against AD suitable for humans remains a longway off (Chapman, Nature, 2000; 408:915-916). The principal hurdlesreside in the extensive work necessary to design and formulate animmunogenic composition that is useful in humans before a practicablevaccine for AD can be achieved. Some of the issues that rely onexperimental data for guidance are: (1) What is the specific target sitefor antibody recognition within the Aβ? (2) In what form should theimmunogen be presented? (3) What other sites need to be included beforean immunogen is achieved that will elicit a therapeutic level ofantibody? (4) What is an effective vaccine delivery system employing aclinically acceptable adjuvant for humans?

A major gap exists between what has been disclosed in the literature andwhat remains to be done. What is the suitable specific target site(i.e., the polymerized Aβ₁₋₄₂ plaque or the monomeric soluble Aβ₁₋₄₂peptide) and how the specific site is to be engineered for immunologicalintervention. In spite of some 5,000 publications on Aβ₁₋₄₂ over thepast decade, the amyloid cascade hypothesis is hotly debated and theissue: the form in which Aβ₁₋₄₂ should be used for intervention remainscontentious. At the heart of the problem, argued by Terry andcolleagues, is the weak correlation between fibrillar amyloid load andmeasures of neurological dysfunction (The Neuropathology of AlzheimerDisease and the Structure Basis of its Alterations, Ed. by Terry et al.,Alzheimer Disease, p 187-206, Lippincott Williams and Wilkins, 1999).

In AD patients, amyloid deposits often form at a distance from the siteof neuron damage. The best correlation with pathological dementia isloss of synaptic terminals. However, the loss of synaptic terminalscorrelates poorly with amyloid load. If the manifestations of diseasecorrelate weakly with amyloid load, then what is the role of Aβ? Thearticle by Klein et al, titled “Targeting small Aβ₁₋₄₂ oligomers: thesolution to an Alzheimer's disease conumdrum?” (Trends in Neurosciences,2001; 24:219-224) suggests that fibrils are not the only toxic form ofAβ, and perhaps not the neurotoxin that is most relevant to AD. Smalloligomers and protofibrils, also termed as Aβ₁₋₄₂ derived diffusibleligands (ADDLs), may also have potent toxic neurological activity.

An AD vaccine for successful immunological intervention will require animmunogen designed to elicit site-directed high affinity antibodies thatbind to the senile plaques in the brain tissue to accelerate theclearance of the plaque by the Glial cells, and immunoneutralize thesoluble Aβ-derived toxins.

The problem of raising high affinity site-directed antibodies againstpoorly immunogenic site-specific peptides have been known for decades.Immunologists and vaccinologists often resort to the classical hapten[peptide]-carrier protein conjugate approach as demonstrated in WO99/27944. For the development of a site-directed vaccine against AD,Frenkel et al. attempted immunization against Aβ₁₋₄₂ plaques through“EFRH”-phage administration (Proc Natl Acad. Sci 2000; 97:11455-11459,WO 01/18169) as mentioned above.

The approaches: using Aβ₁₋₄₂ peptide aggregate or Aβ₁₋₄₂ peptidefragment-carrier protein conjugates (WO99/27944) and using filamentousphage displaying “EFRH peptide” as the agents to induce immune responsesagainst an amyloid deposit in a patient, are cumbersome and ineffective.For example, after the fourth immunization of 1011 phages displaying theEFRH epitope, >95% of the antibodies in the guinea pig immune sera areagainst the phages. Only a small population (<5%) of the antibodies isagainst the soluble Aβ₁₋₄₂ peptide (Frenkel et al., Vaccine 2001,19:2615-2619, WO 01/18169).

Less cumbersome methods were described in EP 526,511 and WO 99/27944,which disclosed the administration of Aβ₁₋₄₂ peptide to treat patientswith pre-established AD and the administration of Aβ₁₋₄₂ or otherimmunogens to a patient under conditions that generate a “beneficial”immune response in the AD patient. However, a review of WO99/27944 showthat there are major deficiencies in the vaccine design disclosedtherein.

In particular, the problem lies in the lack of a pharmaceuticallyacceptable and effective vaccine delivery system. WO99/27944 disclosedAβ₁₋₄₂ or active fragments of Aβ₁₋₄₂ conjugated to a carrier moleculesuch as cholera toxin as the active vaccine component. See page 4 of WO99/27944. Although page 5 taught that a pharmaceutical compositioncomprising the immunogen should be free of Complete Freund's Adjuvant[CFA], the only examples showing the efficacy of the Aβ₁₋₄₂ vaccine forthe treatment of AD in transgenic mice employed large doses ofaggregated Aβ₄₂ peptide in CFA. Despite repetitive recital of preferredadjuvants that are to be used with the disclosed immunogenic agents toenhance the immune response, experimental data showed that only theformulations employing CFA/ICFA provided a sufficiently high titer ofantibodies. See, page 25 of WO 99/27944. In example 1, the prophylacticefficacy of Aβ₁₋₄₂ against AD was demonstrated in PDAPP mice. However,the formulations administered consist a dose of 100 μg per mouse ofaggregated Aβ42 emulsified in Complete Freund's Adjuvant [CFA] (p 34 ofWO 99/27944) followed by multiple booster doses of the same Aβ₁₋₄₂peptide emulsified in Incomplete Freund's Adjuvant. In Example IX, theimmune responses in mice to different adjuvants were studied. When theadjuvants: MPL, Alum, QS21, and CFA/ICFA were used with the purportedlypotent immunogen AN1792 (i.e., aggregated human Aβ₄₂), the level ofantibodies to Aβ₁₋₄ were reduced at a statistically significant level incomparison to mice that received the CFA/ICFA vaccines. See, Table 9,and pages 59-64 of WO 99/27944.

In the case where Aβ₁₋₄₂ peptide fragments were used (human Aβ₁₋₄₂peptides of amino acids 1-5, 1-12, 13-28, and 33-42), each fragment wasconjugated to sheep anti-mouse IgG as the protein carrier. In a laterdisclosure, the efficacy of antibodies to Aβ peptide fragments couldonly be shown by passive immunization with monoclonal antibodies (Bardet al., Nature Medicine 2000; 6:916-919). The efficacy of thesefragments conjugated to sheep anti-mouse IgG was not shown. Therefore,the only immunogen shown to be effective was the aggregated Aβ₁₋₄₂peptide in CFA/ICFA.

Up to the present, all of the vaccine formulations shown to be effectiveemployed CFA/IFA as the adjuvant. Peptide immunogens targeting Aβ₁₋₄₂have thus far been prepared by conjugation of the various Aβ₁₋₄₂fragments to sheep anti-mouse immunoglobulin, conjugation of syntheticAβ₁₃₋₂₈ via m-maleimidobenzoyl-N-hydroxysuccinimide ester to anti-CD3antibody, or aggregated Aβ₁₋₄₂ peptide alone. These immunogens, i.e.,Aβ₄₂ peptide alone or Aβ₁₋₄₂ peptide-carrier protein conjugates, wereemulsified with complete Freund's adjuvant for the first immunization,followed by subsequent boosts in incomplete Freund's adjuvant(Johnson-Wood et al., Proc Natl Acad Sci USA, 1997; 94:1550-1555;Seubert et al., Nature, 1992; 359:325-327; Schenk et al., Nature, 1999;400: 173-177; Janus et al., Nature 2000; 408:979-982; and Morgan et al.,Nature, 2000; 408:982-985). The formulations disclosed in WO 99/27944 orothers using CFA and ICFA as adjuvants cause lesions and are too harshfor use in humans. Thus, none of the vaccine compositions for ADdescribed in the prior art are suitable for use in humans.

In summary, despite statements suggesting the potential of Aβ₁₋₄₂peptide for the treatment of AD in view of the previous disclosures ofKline (EP 526,511), no problem solving vaccine formulations were reallyoffered in WO99/27944 to address this key problem.

Another disadvantage with the peptide-carrier protein conjugates andAβ₁₋₄₂ aggregates is that these molecules are highly complex and aredifficult to characterize and it is difficult to develop effectivequality control procedures for the manufacturing process. A furtherdisadvantage is that, Aβ₁₋₄₂ peptide or its fragments are self moleculeswhen administered to humans. Therefore, they are less immunogenic ornon-immunogenic in humans. It is, thus, necessary to develop clinicallyacceptable vaccine formulations for administration in humans.

It is known that promiscuous Th epitopes may be employed to evokeefficient T cell help and may be combined with poorly immunogenic B cellepitopes to provide potent immunogens. Well-designed promiscuous Th/Bcell epitope chimeric peptides have been shown to be useful in elicitingTh responses and resultant antibody responses in most members of agenetically diverse population expressing diverse MHC haplotypes.Promiscuous Th from a number of pathogens, such as measles virus Fprotein and hepatitis B virus surface antigen, are known. Tables 1 and 2lists many of the known promiscuous Th that have been shown to beeffective in potentiating a short poorly immunogenic peptide, thedecapeptide hormone LHRH (U.S. Pat. Nos. 5,759,551, and 6,025,468).

Potent Th epitopes range in size from approximately 15-40 amino acidresidues in length, often share common structural features, and maycontain specific landmark sequences. For example, a common feature of aTh is that it contains amphipathic helices, alpha-helical structureswith hydrophobic amino acid residues dominating one face of the helixand with charged and polar residues dominating the surrounding faces(Cease et al., Proc Natl Acad Sci USA, 1987; 84: 4249-4253). Th epitopesfrequently contain additional primary amino acid patterns such as a Glyor charged residue followed by two to three hydrophobic residues,followed in turn by a charged or polar residue. This pattern defineswhat are called Rothbard sequences. Th epitopes often obey the 1, 4, 5,8 rule, where a positively charged residue is followed by hydrophobicresidues at the fourth, fifth and eighth positions after the chargedresidue. Since all of these structures are composed of commonhydrophobic, charged and polar amino acids, each structure can existsimultaneously within a single Th epitope (Partidos et al., J Gen Virol,1991; 72:1293). Most, if not all, of the promiscuous T cell epitopes fitat least one of the periodicities described above. These features may beincorporated into the designs of idealized artificial Th sites,including combinatorial Th epitopes. With respect to the design ofcombinatorial Th sites, lists of variable positions and preferred aminoacids are available for MHC-binding motifs (Meister et al., Vaccine,1995; 13:581-591). Furthermore, a method for producing combinatorial Thhas been disclosed for combinatorial library peptides termed structuredsynthetic antigen library (Wang et al., WO 95/11998). Thus, the 1, 4, 5,8 rule can be applied together with known combinatorial MHC-bindingmotifs to assign invariant and degenerate positions in a combinatorialTh site, and to select residues for the degenerate sites to vastlyenlarge the range of immune responsiveness of an artificial Th. See,Table 2, WO 99/66957, and WO 95/11998.

Wang et al. (U.S. Pat. No. 5,759,551) suggested the use ofimmunostimulatory elements to render the self protein Amylinimmunogenic. Wang et al. suggested the administration of immunogenicsynthetic amylin peptides as vaccines for the treatment of non-insulindependent diabetes mellitus (NIDDM), an amyloidogenic disease caused byoverproduction of Amylin (column 19, lines 9-39, U.S. Pat. No.5,759,551). Amylin is a 37 amino acid residue peptide hormone producedby the β cells in the islets of Langerhans. Overproduction of Amylinwill result in the deposition of insoluble amyloid leading toamyloidogenic disease in the pancreas. Similar to the overproduction ofAmylin, overproduction of the Aβ₁₋₄₂ peptide will lead to the depositionof insoluble amyloid in the brain of AD patients. However, there islimited sequence homology between Amylin and the Aβ₁₋₄₂ peptide. Only ashort stretch of amino acids residues, VGSN, of Amylin32-35 correspondsto Aβ₂₄₋₂₇. Antibodies produced against the Amylin peptide are notexpected to be cross reactive to soluble Aβ₁₋₄₂ peptides nor acceleratethe clearance of amyloid plaques in the brain in view of the studies bySoloman et al. and Schenk et al., which showed that the sequence EFRH iscritical.

It is the object of the invention to develop an immunogen that willenable the generation of high levels of high affinity antibodies againstthe N-terminal functional site of the Aβ₁₋₄₂ peptide with highcross-reactivity to the senile plaques in the brain of AD patients. Theantibodies generated by binding to the Aβ₁₋₄₂ peptide and the senileplaques is expected to accelerate the clearance of these plaques fromthe brain, promote fibril disaggregation, inhibit fibrillar aggregation,and cause immunoneutralization of the “soluble Aβ-derived toxins” (alsotermed as Aβ-derived diffusible ligands or ADDLs).

It is a further objective of the present invention to develop a vaccinedelivery vehicle that is suitable for human or veterinary use for theprophylaxis and treatment of Alzheimer's Disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a, 1 b, 1 c, 1 d, 1 e and 1 f are photographs showingImmunoperoxidase staining of serial sections from 2 AD brains, usingAvidin-Biotinylated Antibody Complex (ABC) method with immune andpreimmune sera at 1:100 dilution under 10× magnification. FIGS. 1 a, and1 d show significant binding of antibodies to both senile plaques and Aβplaques (both labelled as “P”) on thioflavine S positive blood vessels(labelled as “BV”). The antibodies were generated in guinea pigs usingAβ₁₋₂₈-εK-MVF Th1-16 (SEQ ID NO:74) prepared in ISA51 water-in-oilemulsion. FIGS. 1 b and 1 e show the cross reactivity of antibodiesraised against the same peptide immunogen in CFA/ICFA. FIGS. 1 c and 1 fshow brain sections using preimmune sera.

FIGS. 2 a, 2 b, 2 c, 2 d, and 2 e are photographs showingImmunoperoxidase staining of serial sections of AD brain with immune andpreimmune sera at 1:100 dilution and under 40× magnification. FIGS. 2 aand 2 d showed that the antibodies in guinea pigs immunized withAβ₁₋₂₈-εK-MVF Th1-16 (SEQ ID NO:74) prepared in ISA51 water-in-oilemulsion strongly stained the plaques (P) forming a pattern of cores.FIG. 2 b is a photograph of the staining pattern of AD brain sectionsusing the same immunogen in CFA/ICFA formulation. The anti-sera reactedpredominantly with plaques on the blood vessels (BV). FIG. 2 c is aphotograph of an AD brain section with preimmune serum and showed nostaining. FIG. 2 e shows the brain section with hyperimmune seragenerated by immunization with Aβ₁₋₂₈ peptide alone in CFA/ICFA showinga surprisingly weak staining pattern despite the strong reactivity withAβ₁₋₂₈ by ELISA.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to an immunogenic composition comprisingsynthetic peptides capable of inducing antibodies against the mainfunctional/regulatory site of the Aβ peptide with high cross-reactivityboth to the soluble Aβ₁₋₄₂ peptide and the plaques in the brain ofAlzheimer's Disease (AD) patients. The immunogenic composition whenadministered to an AD patient or a person predisposed to AD is expectedto accelerate the clearance of amyloid plaques and immunoneutralizationof the soluble Aβ derived toxins in the brain to prevent and treat AD.In particular, a peptide immunogen of this invention comprises a Thepitope selected from the group consisting of SEQ ID NOS: 1-64 and theimmunologically functional analogs thereof linked to a short N-terminalAβ₁₋₄₂ peptide fragment selected from the group consisting of 10 to 28amino acid residues comprising EFRH of the Aβ₁₋₄₂ peptide, SEQ ID NO:65,or an immunologically functional analog of the Aβ₁₋₄₂ peptide fragment.Preferably the Aβ₁₋₄₂ peptide fragment is selected from the group SEQ IDNOS: 66-69 or a immunologically functional analogs thereof.

The present invention further provides an immunogenic compositioncomprising an immunologically effective amount of a peptide compositionin a pharmaceutically acceptable vaccine formulation comprising anadjuvant or emulsifier selected from the group consisting of liposyn,saponin, squalene, L121, emulsigen monophosphyryl lipid A (MPL),polysorbate 80, QS21, Montanide ISA51, ISA35, ISA206 and ISA 720 as wellas the other efficacious adjuvants and emulsifiers.

The present invention further provides a method for the induction ofaccelerated clearance of amyloid plaques and immunoneutralization of thesoluble Aβ-derived toxins in the brain to prevent and treat AlzheimerDisease in a mammal by administering one or more of the immunogenicpeptides to the mammal for a time and under conditions sufficient toinduce antibodies directed against the functional/regulatory site of theAβ₁₋₄₂ peptide. A typical example of a vaccine of the present inventionis a peptide composition comprising 5-1000 μg of the peptide immunogenin a vaccine formulated as a water in oil emulsion in a pharmaceuticallyacceptable adjuvant and/or carrier. A typical method of administeringthe vaccine is to inject intramuscularly the vaccine formulation at0.5-2 mL per dose on an immunization schedule of 0, 4, and 8 weeksintervals.

Yet another aspect of the invention relates to an immunogenic syntheticpeptide of about 30 to about 60 amino acids consisting of a helper Tcell (Th) epitope, linked to an N-terminal Aβ₁₋₄₂ peptide fragmentselected from the group consisting of 10 to 28 amino acids with eachfragment comprising amino acid residue 1 of the Aβ₁₋₄₂ peptide. See SEQID NO:65 wherein D, Aspartic acid, is designated as amino acidresidue 1. Preferably the N terminal Aβ₁₋₄₂ peptide fragment is selectedfrom the group SEQ ID NOs: 66-69 or a peptide analog of N-terminalfragment of Aβ₁₋₄₂ peptide. Optionally, amino acid spacers to separatethe immunogenic domains may be included. The immunogenic domain elementsseparated by spacers can be covalently joined in any order provided thateither the immunoreactivity of the peptide hapten is substantiallypreserved or that immunoreactivity to the N-terminal Aβ peptidefragment, soluble Aβ₁₋₄₂ peptide, and the plaques is generated.

An important factor affecting immunogenicity of a synthetic peptide foran N-terminal Aβ₁₋₄₂ fragment immunogen is its presentation to theimmune system by T helper cell epitopes (Th). Such Th is most reliablysupplied to the peptide immunogen by foreign Th epitopes placed on aseparate Th peptide domain element that is extrinsic to the target Aβpeptide. Such peptide immunogens may be produced as hybrid polypeptidesby recombinant DNA expression. They may also be more simply and lessexpensively supplied as a synthetic peptide immunogen comprising thetarget hapten B cell site from Aβ peptide and T-helper epitopes (Th)appropriate for the host. Such peptides react with helper T-cellreceptors and the class II MHC molecules, in addition to antibodybinding sites (Babbitt et al., Nature, 1985; 317:359) and thus stimulatea tightly site-specific antibody response to the target antibody bindingsite. Previously such Th was supplied for workable Aβ₁₋₄₂ peptideimmunogens by Th intrinsic to aggregated full length Aβ peptide (WO99/66957; WO 1999/27944; Janus et al., 2000, Morgan et al., 2000) andcan be supplied by carrier protein. A wholly synthetic peptide immunogenenjoys the following advantages over Aβ₁₋₄₂ peptide aggregates, carrierconjugates and recombinant polypeptides in that the product ischemically defined for easy quality control. The synthetic peptideimmunogen is stable. No elaborate downstream processing nor an elaboratemanufacturing facility is needed. The immune response is site-specificand focused on the Aβ target and not the carrier. Thus, undesirableresponses such as epitopic suppression are avoided.

Immunogenicity of synthetic N-terminal functional-site directed Aβpeptide immunogens can be optimized by (1) combining N-terminal Aβ₁₋₄₂peptide fragment with selected foreign promiscuous Th sites to which themajority of a population are responsive; and (2) combining Aβ peptidefragment with Th whose repertoire is enlarged through combinatorialchemistry, and thereby accommodate to the variable immune responsivenessof a genetically diverse population.

It has been found that peptides composition of the present invention areeffective in stimulating the production of antibodies against the mainfunctional/regulatory site of the Aβ peptide, with cross-reactivities tothe soluble Aβ₁₋₄₂ and the plaques in the brains of AD patients. Basedon the immunogenicity data obtained in guinea pigs and baboons, and thedata obtained from the immunoperoxidase staining of the amyloid plaquespresent in human AD brain sections by the specific immune sera obtained,it is expected that the peptide immunogens of the present inventionformulated appropriately are effective in humans. It is to be noted thatthe data obtained in baboons are particularly significant in that thisis a species whose immune response closely resemble those of humans.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to a novel peptide composition for thegeneration of high titer polyclonal antibodies with specificity for themain functional/regulatory site of the Aβ peptide, withcross-reactivities to the soluble Aβ₁₋₄₂ and the plaques in the brain ofAlzheimer Disease (AD) patients. The antibodies generated by the peptidecomposition are highly site-specific and bind to the Aβ peptides and toamyloids plaques in the brain. Thus, the present invention provides aneffective method for accelerating the clearance of amyloid plaques andimmunoneutralization of soluble Aβ derived toxins in the brains for theprevention and treatment of AD.

N-terminal Aβ₁₋₄₂ peptide fragments selected from the group consistingof 10 to 28 amino acids wherein each fragment comprises EFRH of theAβ₁₋₄₂ peptide (SEQ ID NO:65), are short linear peptides which, bythemselves are non-immunogenic. The short Aβ₁₋₄₂ peptide fragments canbe immuno-potentiated by chemical coupling to a carrier protein, forexample, keyhole limpet hemocyanin (KLH) or by fusion to a carrierpolypeptide through recombinant DNA expression, for example, hepatitis Bsurface antigen. The deficiency of such “Aβ peptide(s)-carrier protein”vaccines is that a major portion of antibodies generated arenon-functional antibodies directed against the carrier protein.

The immunogens of the present invention are wholly synthetic peptideimmunogens comprising N-terminal fragment of Aβ₁₋₄₂ peptide of 10 to 28amino acids with each fragment comprising EFRH of the Aβ₁₋₄₂ peptidecovalently linked to promiscuous Th epitopes selected from the groupconsisting of SEQ ID NOs: 1 to 64. The immunogens of the inventionelicit the production of site-specific antibodies which bind to theAβ₁₋₄₂ peptide and its aggregates and are cross reactive with amyloidplaques in the brain to provide for accelerated clearance of amyloidplaques and immunoneutralization of the soluble Aβ-derived toxins in thebrain. Thus, the immunogen of the present invention is useful inpreventing and treating AD.

The helper T cell epitopes (Th) useful in the invention comprisemultiple class II MHC binding motifs. Specific examples of Th covalentlylinked to an N-terminal Aβ₁₋₄₂ peptide fragment are provided. Theresults of anti-sera from animals immunized with the immunogen peptidesof the present invention demonstrate that potent site-directed Aβpeptide reactive antibodies are generated, in a genetically diverse hostpopulation.

Generally, the synthetic immunogenic peptides of the present inventionare approximately 20 to 100 amino acids long and comprise:

-   -   (i) a helper T cell (Th) epitope selected from the group        consisting of SEQ ID Nos: 1 to 64;    -   (ii) an N-terminal fragment of Aβ₁₋₄₂ peptide from about 10 to        about 28 amino acid residues wherein each fragment comprises        EFRH of the Aβ₁₋₄₂ peptide; and    -   (iii) optionally a spacer consisting of at least an amino acid        to separate the immunogenic domains.

Preferably, the N terminal fragment of the Aβ₁₋₄₂ peptide is selectedfrom the group consisting of SEQ ID NOS: 66-69 and an immunologicallyeffective analog thereof. The Th peptide is covalently attached toeither the N- or C-terminus of the target N-terminal fragment of Aβ₁₋₄₂peptide optionally with a spacer (e.g., Gly-Gly, ε-N Lys).

The peptide immunogen of this invention is represented by one of thefollowing formula:

(A)_(n)-(N-terminal fragment of Aβ₁₋₄₂ peptide)-(B)_(o)—(Th)_(m)—X; or

(A)_(n)-(Th)_(m)—(B)_(o)—(N-terminal fragment of Aβ₁₋₄₂ peptide)-X;

wherein

-   -   each A is independently an amino acid;    -   each B is a linking group selected from the group consisting of        an amino acid, gly-gly, (α, ε-N)lys, Pro-Pro-Xaa-Pro-Xaa-Pro        (SEQ ID NO:73);    -   Each Th comprise an amino acid sequence that constitutes a        helper T cell epitope, or an immune enhancing analog or segment        thereof;    -   (N-terminal fragment of Aβ₁₋₄₂ peptide) is a synthetic peptide B        cell target site antigen and is a fragment of about 10 to about        28 amino acid residues wherein each fragment comprises EFRH of        the Aβ₁₋₄₂ peptide or an immunologically functional analog        thereof;    -   X is an α-COOH or α-CONH₂ of an amino acid;    -   n is from 0 to about 10;    -   m is from 1 to about 4; and    -   o is from 0 to about 10.

The peptide immunogen of the present invention comprises from about 20to about 100 amino acid residues, preferably from about 25 to about 60amino acid residues. Preferably, the (N-terminal fragment of Aβ₁₋₄₂peptide) is selected from the group consisting of SEQ ID Nos: 66-69 andpreferably the Th epitope is selected from the group consisting of SEQID NOs: 1, 3, 4, 5, 6, 7, 8, 9, 20, 38-40, 47-51 and 52-54. Preferably,m=1, n=1, and o=1 or 2.

When A is an amino acid, it is a non-naturally occurring or naturallyoccurring amino acid. Non-naturally occurring amino acids include, butare not limited to, ε-N lysine, β-alanine, ornithine, norleucine,norvaline, hydroxyproline, thyroxine, γ-amino butyric acid, homoserine,citrulline and the like. Naturally-occurring amino acids includealanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid,glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine andvaline. When m is greater than one, and two or more of A are aminoacids, then each amino acid may independently be the same or different.(A)n may include a spacer, e.g., Gly-Gly, ε-N Lys.

B is a spacer and is an amino acid which can be naturally occurring orthe non-naturally occurring amino acids as described above. Each B isindependently the same or different. The amino acids of B can alsoprovide a spacer, e.g., Gly-Gly, ε-Lys, or lysine between thepromiscuous Th epitope and the N-terminal fragment of Aβ₁₋₄₂ peptide(e.g., SEQ ID NOs:66-69) or an immunologically functional analogthereof. In addition by physically separating the Th epitope from the Bcell epitope, i.e., the N-terminal fragments of Aβ₁₋₄₂ peptide or itsimmunologically functional analog, the Gly-Gly or ε-Lys spacer candisrupt any artifactual secondary structures created by the joining ofthe Th epitope with an N-terminal fragment of Aβ₁₋₄₂ peptide or itsimmunologically functional analog and thereby eliminate interferencebetween the Th and/or B cell responses. The amino acids of B can alsoform a spacer which acts as a flexible hinge that enhances separation ofthe Th and the N-terminal fragments of Aβ₁₋₄₂ peptide. Examples ofsequences encoding flexible hinges are found in the immunoglobulin heavychain hinge region. Flexible hinge sequences are often proline rich. Oneparticularly useful flexible hinge is provided by the sequencePro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO:77), where Xaa is any amino acid, andpreferably aspartic acid. The conformational separation provided by theamino acids of B permits more efficient interactions between thepresented peptide immunogen and the appropriate Th cells and B cells toenhances the immune responses to the Th epitope and theantibody-eliciting epitope or their immunologically functional analogs.

Th is a sequence of amino acids (natural or non-natural amino acids)that comprises a Th epitope. A Th epitope may be a continuous ordiscontinuous epitope. In a discontinuous Th epitope, not every aminoacid of Th is necessary. A Th epitope, or an analog or fragment thereof,is capable of enhancing or stimulating an immune response to theN-terminal fragment of Aβ₁₋₄₂ peptide. Th epitopes that areimmunodominant and promiscuous are highly and broadly reactive acrossanimal and human populations with widely divergent MHC types (Partidoset al., 1991; U.S. Pat. No. 5,759,551). The Th epitope of the subjectpeptides is about 10 to about 50 amino acids, preferably from about 10to about 30 amino acids. When multiple Th epitopes are present (i.e.,m≧2), each Th epitope may be the same or different. A Th segmentcomprises a contiguous portion of a Th epitope that is sufficient toenhance or stimulate an immune response to the N-terminal fragment ofAβ₁₋₄₂ peptide.

Th epitopes of the present invention include those derived from foreignpathogens including but not limited to those exemplified in Table 1 (SEQID Nos:1-21). Further, Th epitopes include idealized artificial Th andartificial idealized combinatorial Th disclosed in WO 99/66957 andlisted here in Table 2 as SEQ ID Nos 22-64. Peptides comprisingcombinatorial Th are produced simultaneously in a single solid-phasepeptide synthesis in tandem with the N-terminal fragment of Aβ₁₋₄₂peptide, A and B. The Th epitopes also include immunologicallyfunctional analogs thereof, having conservative substitutions,additions, deletions and insertions therein of from one to about 10amino acid residues as long as the Th-stimulating function has not beenessentially modified.

In the synthetic peptides of this invention, the Th epitope iscovalently attached through a spacer B to either the N terminus or Cterminus of the N-terminal fragment of Aβ₁₋₄₂ peptide or animmunologically functional analog thereof. An immunologically functionalanalog of the N-terminal fragment of Aβ₁₋₄₂ peptide may compriseconservative substitutions, additions, deletions, or insertions of fromone to about four amino acid residues as long as immune responses thatare crossreactive with the Aβ₁₋₄₂ peptides are elicited. Theconservative substitutions, additions, and insertions can beaccomplished with natural or non-natural amino acids as defined above.

The preferred peptide immunogens of this invention are those comprisingthe N-terminal fragment of the Aβ₁₋₄₂ peptide fragments selected fromthe group consisting of SEQ ID NOs: 66-69 or an immunologicallyfunctional analog thereof; a spacer (e.g., Gly-Gly, ε-Lys); a Th epitopeselected from the group consisting of an HBs Th (SEQ ID NO:1); HBc Th(SEQ ID NO:20); MVF Th (SEQ ID NOS:8, 9); PT Th (SEQ ID NOs:4, 5, 7), TTTh (SEQ ID NOs:3, 4, 6); CT Th (SEQ ID NOs:12, 21); DT Th (SEQ ID NO:13,14), MVF Th derived artificial Th selected from the group consisting ofSEQ ID Nos:38-40, 47-51); HBV Th derived artificial Th selected from thegroup consisting of SEQ ID NOS: 52-54. See Tables 1 and 2.

Peptide compositions which contain a cocktail of the subject peptideimmunogens with two or more Th epitopes may enhance immunoefficacy in abroader population and thus provide an improved immune response to theAβ₁₋₄₂ peptides and their fragments.

The peptide immunogens of this invention can be made by chemicalsynthesis methods which are well known to the ordinarily skilledartisan. See, for example, Fields et al., Chapter 3 in SyntheticPeptides: A User's Guide, ed. Grant, W. H. Freeman & Co., New York,N.Y., 1992, p. 77. Hence, peptides can be synthesized using theautomated Merrifield techniques of solid phase synthesis with the α-NH2protected by either t-Boc or F-moc chemistry using side chain protectedamino acids on, for example, an Applied Biosystems Peptide SynthesizerModel 430A or 431. Preparation of peptide constructs comprisingcombinatorial library peptides for Th epitopes can be accomplished byproviding a mixture of alternative amino acids for coupling at a givenvariable position. After complete assembly of the desired peptideimmunogen, the resin is treated according to standard procedures tocleave the peptide from the resin and deblock the functional groups onthe amino acid side chains. The free peptide is purified by HPLC andcharacterized biochemically, for example, by amino acid analysis or bysequencing. Purification and characterization methods for peptides arewell known to one of ordinary skill in the art.

The immunogen of the present invention may also be prepared as abranched polymer by synthesis of the desired peptide construct directlyonto a branched poly-lysyl core resin (Wang, et al., Science, 1991;254:285-288).

Alternatively, the longer synthetic peptide immunogens can besynthesized by well known recombinant DNA techniques. Such techniquesare provided in well-known standard manuals with detailed protocols. Toconstruct a gene encoding a peptide of this invention, the amino acidsequence is reverse translated to obtain a nucleic acid sequenceencoding the amino acid sequence, preferably with codons that areoptimum for the organism in which the gene is to be expressed. Next, asynthetic gene is made, typically by synthesizing oligonucleotides whichencode the peptide and any regulatory elements, if necessary. Thesynthetic gene is inserted in a suitable cloning vector and transfectedinto a host cell. The peptide is then expressed under suitableconditions appropriate for the selected expression system and host. Thepeptide is purified and characterized by standard methods.

The efficacy of the peptide composition of the present invention can beestablished by injecting an animal, for example, guinea pigs, with animmunogenic composition comprising peptides of the invention. See, Table4, SEQ ID NOS:70-75. The humoral immune response to the N-terminalfragment of Aβ₁₋₄₂ peptide and the soluble Aβ₁₋₄₂ peptide are monitored.A detailed description of the procedures used is provided in theExamples herein below.

Another aspect of this invention provides a peptide compositioncomprising an immunologically effective amount of one or more of thepeptide immunogens of this invention in a pharmaceutically acceptabledelivery system. Accordingly, the subject peptide composition can beformulated as a vaccine using pharmaceutically acceptable adjuvants,carriers or other ingredients routinely employed in the formulation ofvaccines. Among the ingredients that can be used in this invention areadjuvants or emulsifiers including alum, liposyn, saponin, squalene,L121, emulsigen monophosphyryl lipid A (MPL), polysorbate 80, QS21,Montanide ISA51, ISA35, ISA206 and ISA 720 as well as the otherefficacious adjuvants and emulsifiers. The composition may be formulatedfor immediate release or sustained release. The composition may also beformulated for induction of systemic immunity, e.g., by entrapment in orcoadministration with microparticles. Such formulations are readilyavailable to one of ordinary skill in the art.

The immunogens of the present invention can be administered via anyconventional route, such as subcutaneous, oral, intramuscular,parenteral or enteral route. The immunogens can be administered in asingle dose or in multiple doses. A suitable immunization schedule isreadily determined and available to one of ordinary skill in the art.

The peptide composition of the present invention comprises an effectiveamount of one or more of the peptide immunogens of the present inventionand a pharmaceutically acceptable carrier. Such a composition in asuitable dosage unit form generally contains about 0.25 μg to about 500μg of the immunogen per kg body weight. When delivered in multipledoses, the effective amount may be conveniently divided per dosage unit.For example, an initial dose, e.g. 0.0025-0.5 mg per kg body weight;preferably 1-50 μg per kg of body weight of the peptide immunogen is tobe administered by injection, preferably intramuscularly, followed byrepeat (booster) doses of a similar amount. Dosage will depend on theage, weight and general health of the subject as is well known in thevaccine and therapeutic arts.

The immune response of the synthetic Aβ₁₋₄₂ peptide immunogens can beimproved by delivery through entrapment in or on biodegradablemicroparticles of the type described by O'Hagan et al. (Vaccine, 1991;9: 768-771). The immunogens can be encapsulated with or without anadjuvant in biodegradable microparticles, to potentiate immuneresponses, and to provide time-controlled release for sustained orperiodic responses, and for oral administration, (O'Hagan et al., 1991;and, Eldridge et al., 1991; 28: 287-294).

The following examples are provided to illustrate the invention. Thescope of the invention is not to be limited to the specific peptideimmunogens and compositions provided. The examples demonstrate that thepeptide immunogens of the present invention are useful for elicitingsite-directed antibodies to both Aβ₁₋₁₀ and Aβ₁₋₁₄ fragments as well ascross-reactive antibodies to soluble Aβ₁₋₄₂ peptides as early as 4 weeksafter the initial immunization.

EXAMPLE 1 Typical Methods to Synthesize Aβ Peptide Immunogens of thePresent Invention

Peptide immunogens listed in Table 4 (SEQ ID NOS:70-76) were synthesizedindividually by the Merrifield solid-phase synthesis technique onApplied Biosystems automated peptide synthesizers (Models 430, 431 and433A) using Fmoc chemistry. Preparation of peptide immunogens comprisinga combinatorial library Th, i.e., idealized artificial Th site such asMvF derived Th1-8 (SEQ ID NOs:38-40), can be accomplished by providing amixture of the desired amino acids for chemical coupling at a givenposition as specified in the design. After complete assembly of thedesired peptide, the resin was treated according to standard procedureusing trifluoroacetic acid to cleave the peptide from the resin anddeblock the protecting groups on the amino acid side chains. Thecleaved, extracted and washed peptides were purified by HPLC andcharacterized by mass spectrometry and reverse phase HPLC.

EXAMPLE 2 Evaluation of the Immunogenicity of the Aβ Peptide Immunogensof the Present Invention

Aβ-derived peptide immunogens were evaluated on groups of guinea pigs asspecified by the experimental immunization protocol outlined below andby serological assays for determination of immunogenicity.

-   -   Standard Experimental Design:    -   Immunogens: (1) individual peptide immunogen; or        -   (2) a mixture of equal molar peptide immunogens as specified            in each example.    -   Dose: 100 μg in 0.5 mL per immunization unless otherwise        specified    -   Route: intramuscular unless otherwise specified    -   Adjuvants: Complete Freund's Adjuvant (CFA)/Incomplete Adjuvant        (IFA); or other water in oil emulsions otherwise specified.        CFA/IFA groups received CFA week 0, IFA in subsequent weeks.    -   Dose Schedule: 0, 3, and 6 weeks or otherwise specified.    -   Bleed Schedule: weeks 0, 5, 8 or otherwise specified    -   Species: Duncan-Hartley guinea pigs or otherwise specified    -   Assay: Specific ELISAs for each immune serum's anti-peptide        activity. The solid phase substrate was the Aβ peptide fragment        e.g. Aβ₁₋₁₄ or full length Aβ₁₋₄₂ (SEQ ID NOs: 67 and 65). Blood        was collected and processed into serum, and stored prior to        ELISA with the target peptides.

The immunoreactivities of the antibodies elicited against Aβ peptidesand against the soluble Aβ₁₋₄₂ peptides were determined by ELISAs(enzyme-linked immunosorbent assays) using 96-well flat bottommicrotiter plates which were coated with the Aβ₁₋₄₂ peptide fragments,SEQ ID NOs: 67 or 65 as the immunosorbent. Aliquots (100 μL) of thepeptide immunogen solution at a concentration of 5 μg/mL were incubatedfor 1 hour at 37° C. The plates were blocked by another incubation at37° C. for 1 hour with a 3% gelatin/PBS solution. The blocked plateswere then dried and used for the assay. Aliquots (100 μL) of the testimmune sera, starting with a 1:100 dilution in a sample dilution bufferand ten-fold serial dilutions thereafter, were added to the peptidecoated plates. The plates were incubated for 1 hour at 37° C.

The plates were washed six times with 0.05% PBS/Tween® buffer. 100 μL ofhorseradish peroxidase labeled goat-anti-species specific antibody wasadded at appropriate dilutions in conjugate dilution buffer (Phosphatebuffer containing 0.5M NaCl, and normal goat serum). The plates wereincubated for 1 hour at 37° C. before being washed as above. Aliquots(100 μL) of o-phenylenediamine substrate solution were then added. Thecolor was allowed to develop for 5-15 minutes before the enzymatic colorreaction was stopped by the addition of 50 μL 2N H₂SO₄. The A_(492 nm)of the contents of each well was read in a plate reader. ELISA titerswere calculated based on linear regression analysis of the absorbances,with cutoff A_(492 nm) set at 0.5. The cutoff value chosen was rigorouswith the values for diluted normal control samples being less than 0.15.

EXAMPLE 3 Characterization of the Relative Immunogenicities of Aβ₁₋₄₂and its N-Terminal Fragments for Optimization of Design forSite-Directed Aβ Peptide-Based Synthetic Vaccine

To design a total synthetic vaccine that generates a high level of highaffinity antibodies against the Aβ peptides with high cross-reactivityto the soluble Aβ₁₋₄₂ peptides and the plaques in the brain of ADpatients, the relative immunogenicities of Aβ₁₋₄₂ and its N-terminalfragments were characterized initially. In order to determine therelative immunological properties of the various regions within Aβ₁₋₄₂peptide, a mild adjuvant suitable for human use, alum was employed inthe first study. The relative immunogenicities of Aβ₁₋₄₂ peptide and anN-terminal fragment thereof, Aβ₁₋₂₈ were compared. The immunogenicityevaluation was conducted according to procedures described in Example 2.Unexpectedly, Aβ₁₋₂₈ was found to be more immunogenic than the Aβ₁₋₄₂peptide, indicating that there is immunosuppression within C-terminalfragment Aβ₂₉₋₄₂ (Table 5).

Subsequently, the immunogenicity of Aβ₁₋₂₈ was compared to Aβ₁₋₁₄, ashorter N-terminal fragment of Aβ₁₋₄₂. A more potent adjuvant suitablefor human use (Montanide ISA51, Seppic, Paris, FR) was employed for thepreparation of a water-in-oil emulsion for formulating the vaccine.Based on the data obtained as shown in Table 6, the relativeimmunogenicities for the three Aβ peptides (i.e. Aβ₁₋₁₄, Aβ₁₋₂₈ andAβ₁₋₄₂) were ranked Aβ₁₋₂₈>Aβ₁₋₄₂>Aβ₁₋₁₄. Surprisingly, the loss of theC-terminal 14mer from Aβ₁₋₄₂, improved rather than reduced theimmunogenicity. The antibody response against Aβ is primarily directedto the N-terminal region, particularly the Aβ₁₋₁₄ N-terminal fragment asshown by ELISA data (Table 6). However, a further shortening of theAβ₁₋₂₈ fragment from the C-terminal to form the Aβ₁₋₁₄ fragment resultedin a loss in immunogenicity.

The short Aβ₁₋₁₄ fragment contains the main functional/regulatory site,EFRH, located at positions 3-6 of the Aβ₁₋₄₂ peptide as reported bySolomon et al. The blocking of this epitope by antibodies modulates thedynamics of aggregation as well resolubilization of already formedaggregates (Soloman et al., Proc Natl Acad. Sci, 1996; 93:452-455; ProcNatl Aca. Sci, 1997; 94:4109-4112). Most of the anti-Aβ₁₋₂₈ and Aβ₁₋₄₂antibodies are directed against the N-terminal fragment of the Aβ₁₋₄₂peptide containing this epitope (Table 6). However, the Aβ₁₋₁₄ fragmentby itself was poorly immunogenic. The results of this experiment suggestthe presence of an intrinsic Th epitope within the Aβ₁₅₋₂₈ segment. Thisintrinsic Th epitope accounts for the modest immunogenicities of Aβ₁₋₂₈and Aβ₁₋₄₂ peptides in guinea pigs.

The presence of a Th epitope in the Aβ₁₅₋₂₈ fragment is desirable.However, it is desirable to be able to engineer a more potent immunogenfor a successful human vaccine when faced with the limitation of arestricted human MHC molecule, the number of appropriate doses and thetype of adjuvants permitted for human use. Therefore, we attempted thelinkage of a foreign or extrinsic Th such as that derived from HBV Th(SEQ ID NO: 1) to the C-terminal of the Aβ₁₋₂₈ peptide (SEQ ID NO:66).The extrinsic Th epitope significantly enhanced the immunogenicity ofthe Aβ₁₋₂₈ fragment as shown in Table 6. The antibody response to theengineered immunogen with the Aβ₁₋₂₈ fragment remained directed to thefunctional N-terminal fragment of peptide immunogen (SEQ ID NO: 70)making this construct a better immunogen than the Aβ₁₋₂₈ fragment orAβ₁₋₄₂ fragment alone. This peptide immunogen (SEQ ID NO: 70) representsa peptide immunogen with the formula:

(A)_(n)(N-terminal fragment of Aβ peptide)-(B)_(o)—(Th)_(m)

wherein:

-   -   A is αNH₂, with Aβ₁₋₂₈ being an N-terminal fragment of Aβ₁₋₄₂;    -   B is glycine;    -   Th is a helper T cell epitope derived from a foreign pathogen,    -   HBsAg Th (SEQ ID NO: 1), and wherein n is 1, m is 1 and o is 2.

EXAMPLE 4 Lower Limit of N-Terminal Fragment of Aβ for the Developmentof Aβ Based Synthetic Vaccine for AD

Since the main functional/regulatory site comprising the EFRH residuesis located at positions 3-6 of the Aβ₁₋₄₂ peptide (Soloman et al. ProcNatl Acad. Sci, 1996; 93:452-455; Proc Natl Aca. Sci, 1997;94:4109-4112), it was useful to explore the shortest N-terminal fragmentof Aβ₁₋₄₂ peptide as an optimal B cell target site on Aβ forincorporation into the synthetic immunogen of the present invention.

Each of several short non-immunogenic N-terminal fragments of Aβ,Aβ₁₋₁₀, Aβ₁₋₁₂, Aβ₁₋₁₄ along with Aβ₁₋₂₈ was incorporated intoimmunogens designed with a representative idealized artificial Th (SEQID NO:51). Linkage was through an εN-Lys spacer. The engineeredconstructs were formulated with strong adjuvants due to the expected lowimmunogenicity of the short Aβ fragments. The three synthetic constructswere formulated in complete and incomplete Freund's adjuvant and testedfor their immunogenicities based on procedures as described in Example2. As shown in Table 7, all four peptide immunogens were highlyimmunogenic with Log₁₀ ELISA titers in the range from 4.3 to 5.6 (i.e.10^(4.3) to 10^(5.6)) with very high crossreactivities to the fulllength Aβ₁₋₄₂ peptide after only four weeks from the initialimmunization. More importantly, fragments as small as Aβ₁₋₁₀, Aβ₁₋₁₂ andAβ₁₋₁₄ each linked to the idealized artificial Th (SEQ ID NO:51) werefound to be highly immunogenic after linkage to the disclosed artificialTh epitope (Table 7). These peptide immunogens were designed inaccordance with the formula:

(A)_(n)-(N-terminal fragment of Aβ peptide)-(B)_(o)—(Th)_(m)

wherein:

-   -   A is αNH₂, wherein the N-terminal fragment is Aβ₁₋₁₀, Aβ₁₋₁₂,        Aβ₁₋₁₄ or Aβ₁₋₂₈;    -   B is ε-N Lysine, a spacer linked through its epsilon amino group        to the next amino acid;    -   Th is a helper T cell epitope derived from an idealized        artificial Th, MVF Th1-16 (SEQ ID NO:51), wherein n is 1, m is 1        and o is 1.

It was found that further reduction in the length of the N-terminalfragment of Aβ to less than a 10mer would result in more limited, thusundesirable, immunogenicity. It appears that peptides smaller than 10amino acids are problematic for receptor recognition by class II MHCmolecules (Immunology, Fifth edition, ed. Roitt et al., 1998, MosbyInternational Ltd., London, pp 88-89).

Based on this study of Aβ, the useful B cell site derived from Aβ₁₋₄₂should be in the size range of about 10 to about 28 residues.

EXAMPLE 5 Site-Directed Immunoreactivity Targeted by the SyntheticPeptide Immunogen Linked to Artificial Th Epitope

The non-immunogenic N-terminal fragment such as Aβ₁₋₁₄ of Aβ peptide waslinked either through an εN-lysine spacer to an artificial Th peptidedesignated as MVF Th 1-16 (SEQ ID NO:51), or through a standard chemicalcoupling procedure to a conventional carrier protein KLH. The twoimmunogenic constructs were evaluated in guinea pigs for their relative“site-directed” immunogenicities to Aβ peptide and the resultantrespective reactivity of the antibodies towards their respectivecarriers, the artificial Th epitope or the KLH carrier protein,according to the procedures described in Example 2. The short Aβ₁₋₁₄peptide alone as a control immunogen, and the two immunogenic constructswere formulated in a water-in-oil emulsion containing the adjuvantISA51, a formulation that is suitable for human use. As shown in Table8, the N-terminal Aβ₁₋₁₄ fragment by itself is non-immunogenic asexpected. The synthetic immunogen comprising Aβ₁₋₁₄ fragment andartificial Th (SEQ ID NO: 73) was found to be highly immunogenic ineliciting site-directed antibodies to Aβ₁₋₁₄. The antibodies were alsofound to be highly cross-reactive to soluble Aβ₁₋₄₂ peptide as early as4 weeks after the initial immunization (Log₁₀ titers of 4.094 and 4.126for 4 and 6 weeks post initial immunization respectively). When theseAβ-reactive high titer immune sera were tested by ELISA on the MVFTh1-16 peptide (SEQ ID NO 51) coated plate, they were found to benegative (Log₁₀ titer of 0.038 and 0.064 for 4 and 6 weeks post initialimmunization respectively) showing that irrelevant antibodies were notproduced. The data obtained as shown in Table 8 clearly demonstrated thehighly specific site-directed characteristic of the peptide immunogen ofthe present invention.

The immunogen with the carrier protein KLH was found to be highlyimmunoreactive with the conventional peptide-carrier protein conjugate(e.g. Log₁₀ titers of 4.903 and 5.018 for 4 and 6 weeks post initialimmunization respectively). However, the antibodies elicited were onlymoderately crossreactive with the soluble Aβ₁₋₄₂ peptide (e.g. withLog₁₀ titers of 3.342 and 2.736 for 4 and 6 weeks post initialimmunization respectively). This is approximately 10× to 100× less thanSEQ ID NO:73. Unexpectedly, the peptide immunogens of the presentinvention were highly site-directed and focused. Only functionallyimportant antibodies towards the anti-aggregation and disaggregationsites on the N-terminal fragment of the Aβ peptide were generated ratherthan towards irrelevant carrier sites.

EXAMPLE 6 Evaluation of Aβ Peptide Immunogen by Cross-Reactivities toSenile Plaques

Brains of AD patients with plaques and tangles and thioflavine Spositive blood vessels (TSBV) containing amyloid plaques were used forevaluation of cross-reactivities to polymeric senile plaques of theimmune sera raised in guinea pigs and baboons against Aβ peptideimmunogens. Plaques and TSBV reactivities were detected byimmunoperoxidase staining using Avidin-Biotinylated antibody Complex(ABC) method or by immunofluorescence staining using rhodamineconjugated Fab fragment of species specific anti-IgG. All guinea pigsera were tested at a dilution of 1:100 with end point titers determinedfor some of the samples. All baboon sera were tested at a dilution of1:50. The evaluation of the immune and preimmune sera were kindlyperformed under code by Dr. Gaskin as described (Gaskin et al., J. ExpMed. 165:245, 1987).

In FIG. 1, serial cross sections of brains from 2 AD patients wereinitially examined at 10× magnification. Sections (a), (b) and (c) arefrom AD Brain 1 and (d), (e) and (f) are from AD brain 2. Preimmunenormal serum and immune sera from guinea pigs collected at 6 weekspost-initial immunization were tested by immunoperoxidase staining oncryostat sections from AD temporal cortex rich in plaques andneurofilament tangles (NFT). The immune sera used in the first studyshown on slides FIGS. 1 a and 1 d were obtained from animals immunizedwith Aβ₁₋₂₈-εK-MvF Th1-16 (SEQ ID NO:74) prepared in ISA51 water-in-oilemulsion. The results show significant binding to both senile plaquesand amyloid plaques on the thioflavine S positive blood vessels (TSBV).The cross-reactivities of the immune sera raised against the equivalentimmunogen prepared in CFA/ICFA are shown in slides FIGS. 1 b and 1 d.Unexpectedly, in contrast to the results obtained with the vaccineformulated with ISA51, preferential binding to the Aβ₁₋₂₈ plaques on theblood vessels (TSBV) were observed for the sera raised against theCFA/ICFA vaccine. This means that the antibodies elicited by the vaccineformulated with ISA51 is distinguishable from the antibodies raised bythe vaccine formulated in CFA/ICFA. Moreover, the antibodies generatedby the vaccines formulated according to the present invention providedantibodies that have the desired higher cross reactivity to senileplaques in the brain tissue. Preimmune serum gave no staining incorresponding serial sections shown in slides FIGS. 1 c and 1 f.

Further Immunoperoxidase staining of serial cross sections of AD brain 1with preimmune and immune sera at 1:100 dilution are shown in FIGS. 2 ato 2 e at 40× magnification. The sera obtained from animals immunizedwith Aβ₁₋₂₈-εK-MVF Th 1-16 (Seq ID NO:74) prepared in ISA 51water-in-oil emulsion strongly stained the plaques forming a pattern ofcores as shown in slides FIGS. 2 a and 2 d. Again, surprisingly,staining with immune sera prepared against the corresponding CFA/ICFAformulation gave a different staining pattern in that reactivities withplaques were predominantly on the blood vessels as shown in FIG. 2 brather than with the plaques in the brain tissue. Preimmune serum didnot stain the sections as shown in FIG. 2 c. The hyperimmune seragenerated by immunization with Aβ₁₋₄₂ peptide alone in CFA/ICFA, despiteits strong reactivities with Aβ₁₋₂₈ by ELISA, gave a surprisingly weakstaining pattern in the section shown in FIG. 2 e.

Similar immunostaining of AD brain tissue was performed with 11 pooledimmune and preimmune sera obtained from guinea pigs immunized with thevarious vaccine formulations described in Examples 3, 4 and 5. Thesesera were also evaluated for their antibody reactivities with thefunctional-site by Aβ₁₋₁₄ ELISA, and with the soluble Aβ₁₋₄₂ by Aβ₁₋₄₂ELISA (Table 9). In general, parallel trends were found with sera testedin all three assays. As shown in Table 9, the anti-peptide reactivitiesof the pre-immune serum and the sera raised against the short peptideAβ₁₋₁₄ alone formulated in ISA51 water-in-oil emulsion by ELISA were lowand the cross-reactivities to plaques were negligible. Modestreactivities were found with sera from animals vaccinated with Aβ₁₋₂₈peptide alone formulated in Alum and in ISA51, and Aβ₁₋₁₄ conjugated toKLH and formulated in ISA51. Whereas, significant site-directedreactivities to the functional Aβ₁₋₁₄ site, to soluble Aβ, and to theplaques and TSBV in AD patient brain tissue sections were found withsera from animals immunized with synthetic Aβ/Th immunogens of thepresent invention. The results obtained from these studies, therefore,demonstrate excellent and useful immunogenicity of the peptideimmunogens comprising the N-terminal fragment of Aβ₁₋₄₂ having aminoacids from 1-28 to about 1-10, linked to foreign Th epitopes. Moreover,the results showed that the presence of a foreign Th epitope improvesthe immunogenicity of the peptide immunogens of the present invention toa surprising extent. The peptide immunogens of the present invention inclinically acceptable vaccine formulations acceptable to use in humansgenerated antibodies having the desired cross-reactivity to senileplaques in the brain tissues of AD patients.

EXAMPLE 7 The Immunogenicity of Representative Aβ Peptide Vaccines inBaboons as Predictor of Immunotherapeutic Efficacy for AD

A representative synthetic immunogen, Aβ₁₋₂₈-ε-K-MvF Th1-16 (SEQ IDNO:74), formulated in ISA51 water-in-oil emulsion at dose levels of 25μg/0.5 mL, 100 μg/0.5 mL and 400 μg/0.5 mL were given to three baboonsY299, X398, X1198 at 0, 3 and 6 weeks schedule from initialimmunization. Pre-immune sera and sera at weeks 5 and 8 weeks postinitial immunization (wpi) were collected. For comparison, a fourthbaboon X798 was given 100 μg/0.5 mL doses of an equimolar mixture offree peptides Aβ₁₋₂₈ and Aβ₁₋₄₂ formulated in alum, the standardadjuvant approved for human use. Preimmune sera were used as thenegative control.

Sera from all four immunized animals were collected and evaluated fortheir antibody reactivities with the functional site by Aβ₁₋₁₄ ELISA,and for reactivities with soluble Aβ₁₋₄₂ by Aβ₁₋₄₂ ELISA (for seracollected at 0, 5 and 8 wpi). The cross-reactivities of the anti-sera (8wpi only) with the senile plaques and the plaques in thioflavine Spositive blood vessels were evaluated by immunostaining as described inExample 6. Instead of using anti-baboon Ig, the antibody detector usedis an Fab fragment from anti-human IgG that recognizes all humanisotypes and is cross-reactive with baboon IgG.

Parallel trends again were found with sera tested in all three assays.As shown in Table 10, pre-immune sera were negative. Modest ELISAreactivities were found with serum from animal X798 vaccinated withAβ₁₋₂₈ and Aβ₁₋₄₂ formulated in Alum. However, the reactivity of thisserum was weak for the recognition of senile plaques. In contrast,significant site-directed reactivities to the functional-site of Aβ₁₋₁₄,to soluble Aβ₁₋₄₂, and to the plaques and TSBV in AD patient brainsections were found with sera collected at 8 weeks post initialimmunization from animals immunized with the representative compositionof the invention (SEQ ID NO:74) at both the 100 μg/0.5 mL and 400 μg/0.5mL doses formulated with ISA51. The results obtained from this baboonstudy, therefore, demonstrated the usefulness of the immunogen of thepresent invention in a vaccine formulation appropriate for humans. Theimprovement in immunogenicity (10 to 100× increase in specific antibodytiters to the functional-site of Aβ) is very significant in comparisonto the peptide vaccine of the prior art with the immune responsivenessin baboons closely resembling that of humans.

Similarly, a mixture containing two to three synthetic immunogens of thepresent invention can be used for formulation into vaccines at fromabout 25 to 1000 μg per dose to elicit functional anti-Aβ₁₋₁₄ antibodiesin genetically diverse human populations for the prevention andtreatment of AD. Broad immunogenicity in humans is expected due to thepresence of a promiscuous Th epitope in the peptide immunogen of theinvention that provides for achieving broad MHC recognition.

TABLE 1 Pathogen-derived Promiscuous  T Helper Cell Epitopes (Th)Description SEQ ID  of Th Amino Acid Sequence  NO HBs Th^(a)FFLLTRILTIPQSLD 1 PT₁ Th^(a) KKLRRLLYMIYMSGLAVRVHVSKEEQYYDY 2 TT₁ Th^(a)KKQYIKANSKFIGITEL 3 TT₂ Th^(a) KKFNNFTVSFWLRVPKVSASHL 4 PT_(1A) Th^(a)YMSGLAVRVHVSKEE 5 TT₃ Th^(a) YDPNYLRTDSDKDRFLQTMVKLFNRIK 6 PT₂ Th^(a)GAYARCPNGTRALTVAELRGNAEL 7 MVF₁ Th^(a) LSEIKGVIVHRLEGV 8 MVF₂ Th^(a)GILESRGIKARITHVDTESY 9 TT₄ Th^(a) WVRDIIDDFTNESSQKT 10 TT₅ Th^(a)DVSTIVPYIGPALNHV 11 CT Th^(a) ALNIWDRFDVFCTLGATTGYLKGNS 12 DT1 Th^(a)DSETADNLEKTVAALSILPGHGC 13 DT2 Th^(a) EEIVAQSIALSSLMVAQAIPLVGELVDIGF 14AATNFVESC PF Th^(a) DHEKKHAKMEKASSVFNVVNS 15 SM Th^(a) KWFKTNAPNGVDEKHRH16 TraT₁ Th^(a) GLQGKHADAVKAKG 17 TraT₂ Th^(a) GLAAGLVGMAADAMVEDVN 18TraT₃ Th^(a) STETGNQHHYQTRVVSNANK 19 HB_(c50-69) ^(b)SDFFPSVRDLLDTASALYRE 20 CTP₁₁ Th^(c) TINKPKGYVGKE 21 ^(a)US 5,759,551^(b)Ferrari et al., J Clin Invest, 1991; 88:214 ^(c)Stagg et al.,Immunology, 1993; 71:1

TABLE 2 Artificial Idealized Th and Combinatorial Library Idealized Artificial Th Th Identifier Amino Acid SequenceSEQ ID NO a. MVF Th and Th epitopes derived therefrom MVF TMLSEIKGVIVHRLEGV 22 SSAL1 Th1 DLSDLKGL LL HKLDGL 23 EISEIRGI II HRIEGI 24EVSEVRGV VV HRVEGV 25 EFSEFRGF FF HRFEGF 26 MVF Th1-1 ISEIKGVIVHKIEGI 27MTSERTVIVTRMETM 28 LTEIKGVIVHKLEGI 29 MVF Th1-2 ISEIKGVIVHKIEGI 30ITEIRTVIVTRIETI 31 MVF Th1-3 MSEIKGVIVHKLEGM 32 LTEMRTVIVTRMETV 33MVF Th1-4 ISEIKGVIVHKIEGI 34 MVF Th1-5 ITEIRTVIVTRIETI 35 MVF Th1-6MSEMKGVIVHKMEGM 36 MVF Th1-7 LTEIRTVIVTRLETV 37 MVF Th1-8ISISEIKGVIVHKIEGILF 38 ISMTEIRTVIVTRMETMLF 39 ISLSEIKGVIVHKLEGVLF 40MVF Th1-9 ISISEIKGVIVHKIEGILF 41 ISITEIRTVIVTRIETILF 42 MVF Th1-10ISLSEIKGVIVHKLEGMLF 43 ISMTEMRTVIVTRMETVLF 44 MVF Th1-11ISLTEIRTVIVTRLETVLF 45 MVF Th1-12 ISITEIRTVIVTRIETILF 46ISISEIKGVIVHKIEGILF 47 MVF Th1-13 ISITEIRTVIVTRIETILF 48 MVF Th1-14ISMSEMKGVIVHKMEGMLF 49 MVF Th1-15 ISLTEIRTVIVTRLETVLF 50 MVF Th1-16ISITEIKGVIVHRIETILF 51 b. HBsAg Th, Prototype and Derivatives HbsAg-Th1FFLLTRILTIPQSLD 52 HbsAg-Th1-1 KKKFFLLTRILTIPQSLD 53 HbsAg-Th1-2FFLLTRILTIPQSL 54 SSAL2 Th2 KKKLF LL TK L LTLPQSLD 55 RRRIK II TR IITIPLSIR 56 KKKVR VV TK V VTVPISVD 57 KKKFF FF TK F FTFPVSFD 58 KKKLF LLTK L LTLPFSLD 59 HbsAg Th1-3 KKKIITITRIITIITTID 60 HbsAg Th1-4KKKIITITRIITIITTI 61 HbsAg Th1-5 KKKMMTMTRMITMITTID 62 HbsAg Th1-6FITMDTKFLLASTHIL 63 HbsAg Th1-7 KKKFITMDTKFLLASTHIL 64

TABLE 3 Amino Acid Sequences of Aβ₁₋₄₂ Peptides and its N-terminus Fragments SEQ ID NO Amino Acid SequenceSEQ ID NO:65 Aβ₁₋₄₂ DAEFRHDSGYEVHHQKLVFFAEDVGSNK GAIIGLMVGGVVIASEQ ID NO:66 Aβ₁₋₂₈ DAEFRHDSGYEVHHQKLVFFAEDVGSNK SEQ ID NO:67 Aβ₁₋₁₄DAEFRHDSGYEVHH SEQ ID NO:68 Aβ₁₋₁₂ DAEFRHDSGYEV SEQ ID NO:69 Aβ₁₋₁₀DAEFRHDSGY

TABLE 4 SEQ ID Immunogen Amino Acid Sequence NO Aβ₁₋₂₈-GG-HBV ThDAEFRHDSGYEVHHQKLVFFAEDVGSNK-GG-FFLLTRILTIPQSLD 70 Aβ₁₋₁₀-εK-IS-MVFDAEFRHDSGY-εK-ISITEIKGVIVHRIETILF 71 Th1-16 Aβ₁₋₁₂-K-IS-MVFDAEFRHDSGYEV-εK-ISITEIKGVIVHRIETILF 72 Th1-16 Aβ₁₋₁₄-εK-IS-MVFDAEFRHDSGYEVHH-εK-ISITEIKGVIVHRIETILF 73 Th1-16 Aβ₁₋₂₈-εK-IS-MVFDAEFRHDSGYEVHHQKLVFFAEDVGSNK-εK-ISITEIKGVIVHRIETILF 74 Th1-16Aβ₁₋₁₄-εK-MVF DAEFRHDSGYEVHH-εK-ISISEIKGVIVHKIEGILF 75 Th1-9                     T  RT   TR  T 76

TABLE 5 ELISA Titer (Log₁₀) 4 WPI 6 WPI Immunogen Adjuvant GP ID #Aβ₁₋₁₄ Avg. Aβ₁₋₄₂ Avg. Aβ₁₋₁₄ Avg. Aβ₁₋₄₂ Avg. Aβ₁₋₂₈ Alum 1630 1.2442.326 0.878 2.401 0.888 1.966 1.202 2.405 (SEQ ID NO: 66) 1631 3.4083.924 3.044 3.608 Aβ₁₋₄₂ Alum 1634 0.773 1.124 0.680 1.461 1.062 1.7841.203 1.807 (SEQ ID NO: 65) 1635 1.474 2.242 2.505 2.510

TABLE 6 ELISA Titer (Log₁₀) GP 4 WPI 6 WPI Immunogen Adjuvant ID #Aβ₁₋₁₄ Avg. Aβ₁₋₄₂ Avg. Aβ₁₋₁₄ Avg. Aβ₁₋₄₂ Avg. Aβ₁₋₁₄ ISA 51 1658 1.1681.129 1.229 0.975 1.100 1.271 1.285 1.080 (SEQ ID NO: 67) 1659 1.0900.720 1.441 0.874 Aβ₁₋₂₈ ISA51 1632 2.341 2.291 3.656 3.382 2.276 2.7153.359 3.455 (SEQ ID NO: 66) 1633 2.241 3.107 3.153 3.550 Aβ₁₋₂₈-GG-HBVThISA51 1642 4.792 4.612 4.526 4.582 4.548 4.498 4.441 4.261 (SEQ ID NO:70) 1643 4.432 4.637 4.447 4.081 Aβ₁₋₄₂ ISA51 1636 2.724 1.864 3.6032.402 2.286 1.997 3.250 2.873 (SEQ ID NO: 65) 1637 1.004 1.201 1.7072.495

TABLE 7 ELISA Titer (Log₁₀) 4 WPI 6 WPI Immunogen Adjuvant GP ID #Aβ₁₋₁₄ Avg. Aβ₁₋₄₂ Avg. Aβ₁₋₁₄ Avg. Aβ₁₋₄₂ Avg. Aβ₁₋₁₀-εK-MVF CFA/IFA1666 4.293 4.495 4.924 5.087 4.414 4.320 5.180 5.265 Th1-16 1667 4.6965.250 4.225 5.350 (SEQ ID NO: 71) Aβ₁₋₁₂-εK-MVF CFA/IFA 1664 4.577 4.4955.100 4.891 5.320 4.545 6.000 5.278 Th1-16 1665 4.322 4.682 3.700 4.555(SEQ ID NO: 72) Aβ₁₋₁₄-εK-MVF CFA/IFA 1660 3.700 3.285 4.677 5.060 4.5444.683 5.250 5.625 Th1-16 1661 4.764 5.443 4.822 6.000 (SEQ ID NO: 73)Aβ₁₋₂₈-εK-MVF CFA/IFA 1584 3.355 3.201 4.610 4.328 2.743 3.592 4.4874.901 Th1-16 1585 3.707 4.688 3.731 5.155 (SEQ ID NO: 74) 1586 2.5453.685 4.304 5.061

TABLE 8 ELISA Titer (Log₁₀) 4 WPI 6 WPI Th Th peptide peptide ImmunogenAdjuvant GP ID # Aβ₁₋₄₂ Avg. or KLH Avg Aβ₁₋₄₂ Avg. or KLH Avg Aβ₁₋₁₄ISA 51 1658 1.229 0.975 NA NA 1.285 1.080 NA NA (SEQ ID NO: 67) 16590.720 NA 0.874 NA Aβ₁₋₁₄-εK-MVF ISA 51 1662 4.388 4.094 0.006 0.0384.559 4.126 0.065 0.064 Th1-16 1663 3.800 0.070 3.693 0.063 (SEQ ID NO:73) KLH-(C) Aβ₁₋₁₄ ISA 51 1670 3.181 3.342 4.672 4.903 2.625 2.736 4.8765.018 (SEQ ID NO: 67) 1671 3.502 5.133 2.846 5.160

TABLE 9 Immunostaining^(a) of serial frozen ELISA Titer (Log₁₀) sectionsof AD's GP Aβ₁₋₄₂ Aβ₁₋₁₄ brain tissue Vaccine Formulation ID# Avg AvgPlaque TSBV Aβ₁₋₂₈ in Alum 1630 0.878 2.401 1.244 2.326 +1 +4 1631 3.9243.408 Aβ₁₋₂₈ in ISA51 1632 3.686 3.397 2.341 2.291 +3 +5 1633 3.1072.241 Aβ₁₋₂₈-εK-MVF Th1-16 in CFA/IFA 1584 4.610 4.328 3.355 3.201 +4 +6(SEQ ID NO: 74) 1585 4.688 3.707 1586 3.685 2.540 Aβ₁₋₂₈-εK-MVF Th1-16in ISA51 1642 3.603 4.582 2.724 3.510 +4 +6 (SEQ ID NO: 74) 1643 1.2011.004 Aβ₁₋₁₄ in ISA51 1658 1.229 0.975 1.168 1.129 Neg Neg 1659 0.7201.090 Aβ₁₋₁₄-εK-MVF Th1-16 in CFA/IFA 1660 4.677 5.060 3.700 4.232 +4 +6(SEQ ID NO: 73) 1661 5.443 4.764 Aβ₁₋₁₄-εK-MVF Th1-16 in ISA51 16624.388 4.094 3.551 3.285 +4 +6 (SEQ ID NO: 73) 1663 3.800 3.018Aβ₁₋₁₂-εK-MVF Th1-16 in CFA/IFA 1664 5.100 4.891 4.577 4.450 +4 +6 (SEQID NO: 72) 1665 4.682 4.322 Aβ₁₋₁₀-εK-MVF Th1-16 in CFA/IFA 1666 4.9245.087 4.293 4.455 +4 +5 (SEQ ID NO: 71) 1667 5.250 4.696 KLH-(C) Aβ₁₋₁₄in ISA51 1670 3.181 3.342 3.280 3.102 +2 +4 1635 3.502 2.924 NegativeControl <0.5 <0.5 Neg Neg (preimmune serum) ^(a)Serial dilution @ 1:100

TABLE 10 Immunostaining of ELISA Titer (Log₁₀) frozen sections of Aβ₁₋₄₂Aβ₁₋₁₄ AD brain (8 wpi) Group # Vaccine Formulation Dose 0 WPI 5 WPI 8WPI 0 WPI 5 WPI 8WPI Plaques TSBV 1 Aβ₁₋₂₈-εKV-MVF Th1-16  25 μg 0.8942.962 2.736 0.665 1.745 2.706 +2 + 2 in ISA51 100 μg 0.610 2.987 3.6400.794 2.816 4.800 +4 +6 3 (SEQ ID NO: 74) 400 μg 0.696 2.696 4.050 0.5394.250 3.799 +4 +6 4 Aβ₁₋₂₈ + Aβ₁₋₄₂ in Alum 100 μg 0.897 1.963 2.4850.798 0.727 2.850 + + 5 Negative control — — — — — — — Neg Neg

1. A peptide immunogen comprising: (i) a promiscuous helper T cell (Th) epitope of SEQ ID NO: 1; (ii) said promiscuous Th epitope being conjugated to an N-terminal fragment of Aβ₁₋₄₂ peptide consisting of from 10 to 28 amino acid residues wherein each fragment comprises amino acid residue 1 of the Aβ₁₋₄₂ peptide; and (iii) optionally said promiscuous Th epitope is conjugated to the N-terminal fragment of Aβ₁₋₄₂ peptide by means of a spacer consisting of at least an amino acid to separate the Th epitope from the N-terminal fragment of Aβ₁₋₄₂ peptide.
 2. The peptide immunogen of claim 1, wherein the spacer is selected from the group consisting of an amino acid, Gly-Gly, ε-N-Lys, and Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO:77).
 3. The peptide immunogen of claim 1, wherein the spacer is (ε-N)-Lys.
 4. The peptide immunogen of claim 1, wherein the N-terminal fragment of Aβ₁₋₄₂ peptide is SEQ ID NO:
 67. 5. The peptide immunogen of claim 3, wherein the N-terminal fragment of Aβ₁₋₄₂ peptide is SEQ ID NO:
 67. 6. A peptide immunogen, comprising a promiscuous helper T cell (Th) epitope of SEQ ID NO: 1; an N-terminal fragment of Aβ₁₋₄₂ peptide of SEQ ID NO: 67, and an ε-N-Lys spacer.
 7. A peptide immunogen, comprising the following formula: (A)n-(N-terminal fragment of Aβ₁₋₄₂ peptide)-(B)o-(Th)m-X; or (A)n-(Th)m-(B)o-(N-terminal fragment of Aβ₁₋₄₂ peptide)-X; wherein each A is independently an amino acid; each B is a spacer selected from the group consisting of an amino acid, Gly-Gly, ε-N-Lys, and Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO:77); Th comprise an amino acid sequence that constitutes a promiscuous helper T cell epitope SEQ ID NO:1; (N-terminal fragment of Aβ₁₋₄₂ peptide) is 10 to 28 amino acid residues and wherein each fragment comprises amino acid residue 1 of the Aβ₁₋₄₂ peptide; X is an α-COOH or α-CONH₂ of an amino acid; n is 0 to 10; m is 1 to 4; and o is 0 to
 10. 8. The peptide immunogen of claim 7, wherein the spacer is ε-N-Lys.
 9. The peptide immunogen of claim 7, wherein the N-terminal fragment of Aβ₁₋₄₂ peptide is SEQ ID NO:
 67. 10. The peptide immunogen of claim 8, wherein the N-terminal fragment of Aβ₁₋₄₂ peptide is SEQ ID NO:
 67. 11. A composition comprising the peptide immunogen of claim 1 and a pharmaceutically acceptable adjuvant and/or carrier selected from the group consisting of alum, liposyn, saponin, squalene, L121, emulsigen monophosphyryl lipid A (MPL), polysorbate 80, QS21, Montanide ISA51, ISA35, ISA206 and ISA
 720. 12. A composition comprising the peptide immunogen of claim 2 and a pharmaceutically acceptable adjuvant and/or carrier selected from the group consisting of alum, liposyn, saponin, squalene, L121, emulsigen monophosphyryl lipid A (MPL), polysorbate 80, QS21, Montanide ISA51, ISA35, ISA206 and ISA
 720. 13. A composition comprising the peptide immunogen of claim 3 and a pharmaceutically acceptable adjuvant and/or carrier selected from the group consisting of alum, liposyn, saponin, squalene, L121, emulsigen monophosphyryl lipid A (MPL), polysorbate 80, QS21, Montanide ISA51, ISA35, ISA206 and ISA
 720. 14. A composition comprising the peptide immunogen of claim 4 and a pharmaceutically acceptable adjuvant and/or carrier selected from the group consisting of alum, liposyn, saponin, squalene, L121, emulsigen monophosphyryl lipid A (MPL), polysorbate 80, QS21, Montanide ISA51, ISA35, ISA206 and ISA
 720. 15. A composition comprising the peptide immunogen of claim 5 and a pharmaceutically acceptable adjuvant and/or carrier selected from the group consisting of alum, liposyn, saponin, squalene, L121, emulsigen monophosphyryl lipid A (MPL), polysorbate 80, QS21, Montanide ISA51, ISA35, ISA206 and ISA
 720. 16. A composition comprising the peptide immunogen of claim 6 and a pharmaceutically acceptable adjuvant and/or carrier selected from the group consisting of alum, liposyn, saponin, squalene, L121, emulsigen monophosphyryl lipid A (MPL), polysorbate 80, QS21, Montanide ISA51, ISA35, ISA206 and ISA
 720. 17. A composition comprising the peptide immunogen of claim 7 and a pharmaceutically acceptable adjuvant and/or carrier selected from the group consisting of alum, liposyn, saponin, squalene, L121, emulsigen monophosphyryl lipid A (MPL), polysorbate 80, QS21, Montanide ISA51, ISA35, ISA206 and ISA
 720. 18. A composition comprising the peptide immunogen of claim 8 and a pharmaceutically acceptable adjuvant and/or carrier selected from the group consisting of alum, liposyn, saponin, squalene, L121, emulsigen monophosphyryl lipid A (MPL), polysorbate 80, QS21, Montanide ISA51, ISA35, ISA206 and ISA
 720. 19. A composition comprising the peptide immunogen of claim 9 and a pharmaceutically acceptable adjuvant and/or carrier selected from the group consisting of alum, liposyn, saponin, squalene, L121, emulsigen monophosphyryl lipid A (MPL), polysorbate 80, QS21, Montanide ISA51, ISA35, ISA206 and ISA
 720. 20. A composition comprising the peptide immunogen of claim 10 and a pharmaceutically acceptable adjuvant and/or carrier selected from the group consisting of alum, liposyn, saponin, squalene, L121, emulsigen monophosphyryl lipid A (MPL), polysorbate 80, QS21, Montanide ISA51, ISA35, ISA206 and ISA
 720. 